Longitudinal Micro-Computed Tomography Detects Onset and Progression of Pulmonary Fibrosis in Conditional Nedd4-2 Deficient Mice.

OBJECTIVES: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease which is usually diagnosed late in advanced stages. Little is known about the subclinical development of IPF. We previously generated a mouse model with conditional Nedd4-2 deficiency (Nedd4-2-/-) that develops IPF-like lung disease. The aim of this study was to characterize the onset and progression of IPF-like lung disease in conditional Nedd4-2-/- mice by longitudinal micro-computed tomography (CT). METHODS: In vivo micro-CT was performed longitudinally in control and conditional Nedd4-2-/- mice at 1, 2, 3, 4 and 5 months after doxycycline induction. Further, terminal in vivo micro-CT followed by pulmonary function testing and post mortem micro-CT was performed in age-matched mice. Micro-CT images were evaluated for pulmonary fibrosis using an adapted fibrosis scoring system. Histological assessment of lung collagen content was conducted as well. RESULTS: Micro-CT is sensitive to detect onset and progression of pulmonary fibrosis in vivo and to quantify distinct radiological IPF-like features along disease development in conditional Nedd4-2-/- mice. Nonspecific interstitial alterations were detected from 3 months, whereas key features such as honeycombing-like lesions were detected from 4 months onwards. Pulmonary function correlated well with in vivo (r=-0.738) and post mortem (r=-0.633) micro-CT fibrosis scores and collagen content. CONCLUSION: Longitudinal micro-CT enables in vivo monitoring of onset and progression and detects radiologic key features of IPF-like lung disease in conditional Nedd4-2-/- mice. Our data support micro-CT as sensitive quantitative endpoint for preclinical evaluation of novel antifibrotic strategies.

Novel setup for rapid phase contrast CT imaging of heavy and bulky specimens.

This work introduces a novel setup for computed tomography of heavy and bulky specimens at the SYRMEP beamline of the Italian synchrotron Elettra. All the key features of the setup are described and the first application to off-center computed tomography scanning of a human chest phantom (approximately 45 kg) as well as the first results for vertical helical acquisitions are discussed.

Evaluation of classifications of the monopodial bronchopulmonary vasculature using clustering methods.

Mammalian pulmonary arteries divide multiple times before reaching the vast capillary network of the alveoli. Morphological analyses of the arterial branches can be challenging because more proximal branches are likely biologically distinct from more peripheral parts. Thus, it is useful to group the arterial branches into groups of coherent biology. While the generational approach of dichotomous branching is straightforward, the grouping of arterial branches in the asymmetrically branching monopodial lung is less clear. Several established classification methods return highly dissimilar groupings when employed on the same organ. Here, we established a workflow allowing the quantification of grouping results for the monopodial lung and tested various methods to group the branches of the arterial tree into coherent groups. A mouse lung was imaged by synchrotron x-ray microcomputed tomography, and the arteries were digitally segmented. The arterial tree was divided into its individual segments, morphological properties were assessed from corresponding light microscopic scans, and different grouping methods were employed, such as (fractal) generation or (Strahler) order. The results were ranked by the morphological similarity within and dissimilarity between the resulting groups. Additionally, a method from the mathematical field of cluster analysis was employed for creating a reference classification. In conclusion, there were significant differences in method performance. The Strahler order was significantly superior to the generation system commonly used to classify human lung structure. Furthermore, a clustering approach indicated more precise ways to classify the monopodial lung vasculature exist.

Alpine dwarf shrubs show high proportions of nonfunctional xylem: Visualization and quantification of species-specific patterns.

Xylem conductive capacity is a key determinant of plant hydraulic function and intimately linked to photosynthesis and productivity, but can be impeded by temporary or permanent conduit dysfunctions. Here we show that persistent xylem dysfunctions in unstressed plants are frequent in Alpine dwarf shrubs and occur in various but species-specific cross-sectional patterns. Combined synchrotron micro-computed tomography (micro-CT) imaging, xylem staining, and flow measurements in saturated samples of six widespread Ericaceae species evidence a high proportion (19%-50%) of hydraulically nonfunctional xylem areas in the absence of drought stress, with regular distribution of dysfunctions between or within growth rings. Dysfunctions were only partly reversible and reduced the specific hydraulic conductivity to 1.38 to 3.57 ×10-4 m2 s-1 MPa-1 . Decommission of inner growth rings was clearly related to stem age and a higher vulnerability to cavitation of older rings, while the high proportion of nonfunctional conduits in each annual ring needs further investigations. The lower the xylem fraction contributing to the transport function, the higher was the hydraulic efficiency of conducting xylem areas. Improved understanding of the functional lifespan of xylem elements and the prevalence and nature of dysfunctions is critical to correctly assess structure-function relationships and whole-plant hydraulic strategies.

Comparison of propagation-based CT using synchrotron radiation and conventional cone-beam CT for breast imaging.

OBJECTIVES: To evaluate and compare the image quality of propagation-based phase-contrast computed tomography (PB-CT) using synchrotron radiation and conventional cone-beam breast computed tomography (CBBCT) based on various radiological image quality criteria. METHODS: Eight excised breast tissue samples of various sizes and containing different lesion types were scanned using PB-CT at a synchrotron facility and using CBBCT at a university-affiliated breast imaging centre. PB-CT scans were performed at two different mean glandular dose (MGD) levels: standard (5.8 mGy) and low (1.5 mGy), for comparison with CBBCT scans at the standard MGD (5.8 mGy). Image quality assessment was carried out using six quality criteria and six independent medical imaging experts in a reading room with mammography workstations. The interobserver agreement between readers was evaluated using intraclass correlation coefficient (ICC), and image quality was compared between the two breast imaging modalities using the area under the visual grading characteristic curve (AUCVGC). RESULTS: Interobserver agreement between the readers showed moderate reliability for five image criteria (ICC: ranging from 0.488 to 0.633) and low reliability for one criterion (image noise) (ICC 0.307). For five image quality criteria (overall quality, perceptible contrast, lesion sharpness, normal tissue interfaces, and calcification visibility), both standard-dose PB-CT images (AUCVGC 0.958 to 1, p ≤ .05) and low dose PB-CT images (AUCVGC 0.785 to 0.834, p ≤ .05) were of significantly higher image quality than standard-dose CBBCT images. CONCLUSIONS: Synchrotron-based PB-CT can achieve a significantly higher radiological image quality at a substantially lower radiation dose compared with conventional CBBCT. KEY POINTS: • PB-CT using synchrotron radiation results in higher image quality than conventional CBBCT for breast imaging. • PB-CT using synchrotron radiation requires a lower radiation dose than conventional CBBCT for breast imaging. • PB-CT can help clinicians diagnose patients with breast cancer.

Detecting Early Markers of Ventilator-Associated Pneumonia by Analysis of Exhaled Gas.

OBJECTIVES: The detection of microbial volatile organic compounds or host response markers in the exhaled gas could give an earlier diagnosis of ventilator-associated pneumonia. Gas chromatography-ion mobility spectrometry enables noninvasive, rapid, and sensitive analysis of exhaled gas. Using a rabbit model of ventilator-associated pneumonia we determined if gas chromatography-ion mobility spectrometry is able to detect 1) ventilator-associated pneumonia specific changes and 2) bacterial species-specific changes in the exhaled gas. DESIGN: Experimental in vivo study. SETTING: University research laboratory. SUBJECTS: Female New Zealand White rabbits. INTERVENTIONS: Animals were anesthetized and mechanically ventilated. To induce changes in the composition of exhaled gas we induced ventilator-associated pneumonia via endobronchial instillation of either Escherichia coli group (n = 11) or Pseudomonas aeruginosa group (n = 11) after 2 hours of mechanical ventilation. In a control group (n = 11) we instilled sterile lysogeny broth endobronchially. MEASUREMENTS AND MAIN RESULTS: Gas chromatography-ion mobility spectrometry gas analysis, CT scans of the lungs, and blood samples were obtained at four measurement points during the 10 hours of mechanical ventilation. The volatile organic compound patterns in the exhaled gas were compared and correlated with ventilator-associated pneumonia severity. Sixty-seven peak areas showed changes in signal intensity in the serial gas analyses. The signal intensity changes in 10 peak regions differed between the groups. Five peak areas (P_648_36, indole, P_714_278, P_700_549, and P_727_557) showed statistically significant changes of signal intensity. CONCLUSIONS: This is the first in vivo study that shows the potential of gas chromatography-ion mobility spectrometry for early detection of ventilator-associated pneumonia specific volatile organic compounds and species differentiation by noninvasive analyses of exhaled gas.

Insights from in vivo micro-CT analysis: testing the hydraulic vulnerability segmentation in Acer pseudoplatanus and Fagus sylvatica seedlings.

The seedling stage is the most susceptible one during a tree's life. Water relations may be crucial for seedlings due to their small roots, limited water buffers and the effects of drought on water transport. Despite obvious relevance, studies on seedling xylem hydraulics are scarce as respective methodical approaches are limited. Micro-CT scans of intact Acer pseudoplatanus and Fagus sylvatica seedlings dehydrated to different water potentials (Ψ) allowed the simultaneous observation of gas-filled versus water-filled conduits and the calculation of percentage loss of conductivity (PLC) in stems, roots and leaves (petioles or main veins). Additionally, anatomical analyses were performed and stem PLC measured with hydraulic techniques. In A. pseudoplatanus, petioles showed a higher Ψ at 50% PLC (Ψ50 -1.13MPa) than stems (-2.51 MPa) and roots (-1.78 MPa). The main leaf veins of F. sylvatica had similar Ψ50 values (-2.26 MPa) to stems (-2.74 MPa) and roots (-2.75 MPa). In both species, no difference between root and stems was observed. Hydraulic measurements on stems closely matched the micro-CT based PLC calculations. Micro-CT analyses indicated a species-specific hydraulic architecture. Vulnerability segmentation, enabling a disconnection of the hydraulic pathway upon drought, was observed in A. pseudoplatanus but not in the especially shade-tolerant F. sylvatica. Hydraulic patterns could partly be related to xylem anatomical traits.

Synchrotron inline phase contrast µCT enables detailed virtual histology of embedded soft-tissue samples with and without staining.

Synchrotron radiation micro-computed tomography (SRµCT) based virtual histology, in combination with dedicated ex vivo staining protocols and/or phase contrast, is an emerging technology that makes use of three-dimensional images to provide novel insights into the structure of tissue samples at microscopic resolution with short acquisition times of the order of minutes or seconds. However, the high radiation dose creates special demands on sample preparation and staining. As a result of the lack of specific staining in virtual histology, it can supplement but not replace classical histology. Therefore, the aim of this study was to establish and compare optimized ex vivo staining and acquisition protocols for SRµCT-based virtual histology of soft-tissue samples, which could be integrated into the standard workflow of classical histology. The high grade of coherence of synchrotron radiation allows the application of propagation-based phase contrast imaging (PBI). In this study, PBI yielded a strong increase in image quality even at lower radiation doses and consequently prevented any damage to the tissue samples or the embedding material. This work has demonstrated that the improvement in contrast-to-noise ratio by PBI enabled label-free virtual histology of soft-tissue specimens embedded in paraffin to a level of detail that exceeds that achieved with staining protocols.

MÖNCH detector enables fast and low-dose free-propagation phase-contrast computed tomography of in situ mouse lungs.

Due to the complexity of the underlying pathomechanism, in vivo mouse lung-disease models continue to be of great importance in preclinical respiratory research. Longitudinal studies following the cause of a disease or evaluating treatment efficacy are of particular interest but challenging due to the small size of the mouse lung and the fast breathing rate. Synchrotron-based in-line phase-contrast computed tomography imaging has been successfully applied in lung research in various applications, but mostly at dose levels that forbid longitudinal in vivo studies. Here, the novel charge-integrating hybrid detector MÖNCH is presented, which enables imaging of mouse lungs at a pixel size of 25 µm, in less than 10 s and with an entrance dose of about 70 mGy, which therefore will allow longitudinal lung disease studies to be performed in mouse models.

Towards synchrotron phase-contrast lung imaging in patients - a proof-of-concept study on porcine lungs in a human-scale chest phantom.

In-line free propagation phase-contrast synchrotron tomography of the lungs has been shown to provide superior image quality compared with attenuation-based computed tomography (CT) in small-animal studies. The present study was performed to prove the applicability on a human-patient scale using a chest phantom with ventilated fresh porcine lungs. Local areas of interest were imaged with a pixel size of 100 µm, yielding a high-resolution depiction of anatomical hallmarks of healthy lungs and artificial lung nodules. Details like fine spiculations into surrounding alveolar spaces were shown on a micrometre scale. Minor differences in artificial lung nodule density were detected by phase retrieval. Since we only applied a fraction of the X-ray dose used for clinical high-resolution CT scans, it is believed that this approach may become applicable to the detailed assessment of focal lung lesions in patients in the future.

Advantages of breast cancer visualization and characterization using synchrotron radiation phase-contrast tomography.

The aim of this study was to highlight the advantages that propagation-based phase-contrast computed tomography (PB-CT) with synchrotron radiation can provide in breast cancer diagnostics. For the first time, a fresh and intact mastectomy sample from a 60 year old patient was scanned on the IMBL beamline at the Australian Synchrotron in PB-CT mode and reconstructed. The clinical picture was described and characterized by an experienced breast radiologist, who underlined the advantages of providing diagnosis on a PB-CT volume rather than conventional two-dimensional modalities. Subsequently, the image quality was assessed by 11 breast radiologists and medical imaging experts using a radiological scoring system. The results indicate that, with the radiation dose delivered to the sample being equal, the accuracy of a diagnosis made on PB-CT images is significantly higher than one using conventional techniques.

The pitfalls of in vivo imaging techniques: evidence for cellular damage caused by synchrotron X-ray computed micro-tomography.

Synchrotron X-ray computed micro-tomography (microCT) has emerged as a promising noninvasive technique for in vivo monitoring of xylem function, including embolism build-up under drought and hydraulic recovery following re-irrigation. Yet, the possible harmful effects of ionizing radiation on plant tissues have never been quantified. We specifically investigated the eventual damage suffered by stem living cells of three different species exposed to repeated microCT scans. Stem samples exposed to one, two or three scans were used to measure cell membrane and RNA integrity, and compared to controls never exposed to X-rays. Samples exposed to microCT scans suffered serious alterations to cell membranes, as revealed by marked increase in relative electrolyte leakage, and also underwent severe damage to RNA integrity. The negative effects of X-rays were apparent in all species tested, but the magnitude of damage and the minimum number of scans inducing negative effects were species-specific. Our data show that multiple microCT scans lead to disruption of fundamental cellular functions and processes. Hence, microCT investigation of phenomena that depend on physiological activity of living cells may produce erroneous results and lead to incorrect conclusions.

X-ray-based lung function measurement reveals persistent loss of lung tissue elasticity in mice recovered from allergic airway inflammation.

Chronic asthma patients experience difficulties even years after the inciting allergen. Although studies in small animal asthma models have enormously advanced progress in uncovering the mechanisms of inception and development of the disease, little is known about the processes involved in the persistence of asthma symptoms in the absence of allergen exposure. Long-term asthma mouse models have so far been scarce or not been able to reproduce the findings in patients. Here we used a common ovalbumin-induced acute allergic airway inflammation mouse model to study lung function and remodeling after a 4-mo recovery period. We show by X-ray-based lung function measurements that the recovered mice continue to show impaired lung function by displaying significant air trapping compared with controls. High-resolution synchrotron phase-contrast computed tomography of structural alterations and diaphragm motion analysis suggest that these changes in pulmonary function are the result of a pronounced loss in lung elasticity. Histology of lung sections confirmed that this is most likely caused by a decrease in elastic fibers, indicating that remodeling can develop or persist independent of acute inflammation and is closely related to a loss in lung function. Our findings demonstrate that this X-ray-based imaging platform has the potential to comprehensively and noninvasively unravel long-term effects in preclinical mouse models of allergic airway inflammation and thus benefits our understanding of chronic asthma.

Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation.

In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype, and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription, and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord, and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a BM cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13, and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs), as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age, with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

Preclinical evaluation of near-infrared (NIR) fluorescently labeled cetuximab as a potential tool for fluorescence-guided surgery.

The high rate of recurrence in patients with pancreatic ductal adenocarcinoma (PDAC) could be reduced by supporting the surgeons in discriminating healthy from diseased tissues with intraoperative fluorescence-guidance. Here, we studied the suitability of Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), near-infrared (NIR) fluorescently labeled as a new tool for fluorescence-guided surgery. Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux-Alexa-647) and the co-injected control human IgG Alexa Fluor 750 conjugate (hIgG-Alexa-750) was studied over 48 h by NIR fluorescence imaging in mice bearing human orthotopic AsPC-1 and MIA PaCa-2 PDAC tumors. Cetux-Alexa-647, but not the control hIgG-Alexa-750 fluorescence, was specifically detected in vivo in both primary pancreatic tumors with maximum fluorescence intensities at 24 h, and in metastases of AsPC-1 tumors as small as 1 mm. Lifetime analysis and NIR fluorescence microscopy of tumor sections confirmed the binding specificity of Cetux-Alexa-647 to PDAC cells. Comparable results were obtained with Cetuximab conjugated to Alexa Fluor 750 dye (Cetux-Alexa-750). Fluorescence-guided dissection, performed 24 h after injection of Cetuximab conjugated to IRDye 800CW (Cetux-800CW), enabled a real-time delineation of AsPC-1 tumor margins, and small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part was stained with Cetux-800CW. NIR fluorescently labeled Cetuximab may be a promising tool that can be applied for fluorescence-guided surgery to visualize tumor margins and metastatic sites in order to allow a precise surgical resection.

Phase-contrast computed tomography for quantification of structural changes in lungs of asthma mouse models of different severity.

Lung imaging in mouse disease models is crucial for the assessment of the severity of airway disease but remains challenging due to the small size and the high porosity of the organ. Synchrotron inline free-propagation phase-contrast computed tomography (CT) with its intrinsic high soft-tissue contrast provides the necessary sensitivity and spatial resolution to analyse the mouse lung structure in great detail. Here, this technique has been applied in combination with single-distance phase retrieval to quantify alterations of the lung structure in experimental asthma mouse models of different severity. In order to mimic an in vivo situation as close as possible, the lungs were inflated with air at a constant physiological pressure. Entire mice were embedded in agarose gel and imaged using inline free-propagation phase-contrast CT at the SYRMEP beamline (Synchrotron Light Source, `Elettra', Trieste, Italy). The quantification of the obtained phase-contrast CT data sets revealed an increasing lung soft-tissue content in mice correlating with the degree of the severity of experimental allergic airways disease. In this way, it was possible to successfully discriminate between healthy controls and mice with either mild or severe allergic airway disease. It is believed that this approach may have the potential to evaluate the efficacy of novel therapeutic strategies that target airway remodelling processes in asthma.

Functionalized synchrotron in-line phase-contrast computed tomography: a novel approach for simultaneous quantification of structural alterations and localization of barium-labelled alveolar macrophages within mouse lung samples.

Functionalized computed tomography (CT) in combination with labelled cells is virtually non-existent due to the limited sensitivity of X-ray-absorption-based imaging, but would be highly desirable to realise cell tracking studies in entire organisms. In this study we applied in-line free propagation X-ray phase-contrast CT (XPCT) in an allergic asthma mouse model to assess structural changes as well as the biodistribution of barium-labelled macrophages in lung tissue. Alveolar macrophages that were barium-sulfate-loaded and fluorescent-labelled were instilled intratracheally into asthmatic and control mice. Mice were sacrificed after 24 h, lungs were kept in situ, inflated with air and scanned utilizing XPCT at the SYRMEP beamline (Elettra Synchrotron Light Source, Italy). Single-distance phase retrieval was used to generate data sets with ten times greater contrast-to-noise ratio than absorption-based CT (in our setup), thus allowing to depict and quantify structural hallmarks of asthmatic lungs such as reduced air volume, obstruction of airways and increased soft-tissue content. Furthermore, we found a higher concentration as well as a specific accumulation of the barium-labelled macrophages in asthmatic lung tissue. It is believe that XPCT will be beneficial in preclinical asthma research for both the assessment of therapeutic response as well as the analysis of the role of the recruitment of macrophages to inflammatory sites.

The Impact of Strontium Ranelate on Metaphyseal Bone Healing in Ovariectomized Rats.

The following questions were addressed: whether therapy with strontium ranelate (SR) should be continued or interrupted if the fractures occur during SR treatment and whether SR could be applied directly after fracture to improve bone healing. Sprague-Dawley rats (3 month old) were ovariectomized (Ovx, n = 48) or left intact (n = 12). After 8 weeks, a bilateral transverse osteotomy of the tibia metaphysis was created in all rats. Ovx rats were divided into four groups: Ovx; SR applied directly after Ovx until osteotomy (prophylaxis, SR pr, 8 weeks); SR applied after osteotomy (therapy, SR th, 5 weeks); SR applied during the whole experiment (pr + th, 13 weeks). SR dosage was 625 mg/kg body weight/day, administered in the feed. Five weeks later, tibiae were analyzed by biomechanical, histological, micro-CT, and gene expression analyses. The SR pr + th treatment increased total bone mineral density (BMD), bone volume fraction, cortical BMD and volume, callus area and density, serum alkaline phosphatase, tartrate-resistant acid phosphatase mRNA, accelerated osteotomy bridging, and callus formation at weeks 2 and 3 of healing and decreased the osteoprotegerin/receptor activator of nuclear factor kB ligand mRNA ratio. SR th enlarged callus area and improved callus formation during the 5th week of healing. SR pr improved cortical BMD preserving bone after SR discontinuation (5-week rest); the bone healing was not affected. SR content in the tibia metaphysis was the highest in SR pr + th group and was not different between SR pr and SR th. SR has a positive effect on osteoporotic bone healing in rat and SR treatment can be continued after the fracture occurs or applied directly after the fracture.